Discovering smoking-related pathway alterations in urothelial cell carcinoma pathogenesis

Urothelial cell carcinoma (UCC) represents the most common malignancy of the urinary tract. It is estimated that there were 73,500 new cases of UCC and 15,000 deaths for both sexes in the United States in 2012.¹ As the final recipient and reservoir of urine, the urothelium is inevitably exposed to carcinogens present in tobacco, which can create stepwise molecular alterations that eventually lead to transformation of urothelial cells. This concept is supported by epidemiologic studies that state that tobacco consumption is the most important factor for the development of this disease, contributing to approximately 50% of all cases.²

UCC is a heterogeneous disease; 70% of newly diagnosed bladder tumors are non-muscle invasive (NMIBC) and show a much better prognosis compared with those that invade the detrusor muscle (MIBC).³ From the molecular point of view, evidence in the literature supports the existence of two distinct pathogenetic pathways involved in UCC development, corresponding to these two distinct (NMIBC and MIBC) biological and clinical phenotypes. In fact, while disruption to the PI3K-AKT-mTOR pathway and alterations in the tyrosine kinase receptor gene FGFR3 and the oncogene HRAS are associated with NMIBC, the main genetic alterations underlying MIBC involve tumor suppressor genes encoding proteins that regulate cell cycle and apoptosis pathways, including TP53, CDKN2A, CCND1, CDKN1B and RB1.⁴ Recent works have also suggested epigenetic mechanisms like promoter methylation in the pathogenesis of this disease.^5,6

Understanding the multistep accumulation of genetic and epigenetic alterations related to environmental factors in the development and progression of this disease is crucial for the discovery of biomarkers that might be useful in predicting the behavior and prognosis of UCC in individual patients. In an intent to understand the genetic/epigenetic alterations that accumulates in the process of UCC development, the group led by Hoque has developed a very interesting cellular model for smoking-induced UCC.⁷ In this study, SV-40 immortalized normal HUC1 human bladder epithelial cells were continuously exposed to 0.1% cigarette smoke extract (CSE) until transformation occurred. The authors observed morphological alterations and increased cell proliferation after 4 mo of exposure to CSE. After 6 mo the treated cells showed anchorage-independent growth and an increase in the migratory and invasive potential. The observed properties after 6 mo of CSE treatment were not noticeable at 4 mo of treatment, suggesting that some driver gene/genes might alter due to prolonged exposure to tobacco.

In order to assess key molecular alterations occurring in CSE-treated cells, the authors evaluated the expression level of specific genes involved in the PI3K-AKT pathway and found upregulation of AKT1, AKT2, HRAS, RAC1 and downregulation of PTEN, FOXO1, MAPK1 and PDK1 among altered genes. Interestingly, immunohistochemistry for FOXO1 performed on UCC samples showed higher level and frequency of expression in the smokers group compared with non-smokers. In their view, this might reflect the fact that FOXO1 in smokers is subjected to an enhanced phosphorylation by AKT with consequent cytoplasmic translocation. Using genome-wide methylation analysis, the authors also found differentially methylated genes in CSE-treated and untreated HUC1 cell lines. They further confirmed methylation status of MCAM, DCC and HIC1 in CSE-treated and untreated HUC1 cell lines by a complementary approach (QMSP). These findings support that epigenetic alterations are simultaneously related to smoke-associated UCC.

As stated above, P53 represents the most frequently dysregulated gene in UCC, especially in the pathway related to muscle-invasive tumors. However, in this work, translational and transcriptional levels of P53 were unchanged after 6 mo of CSE treatment. In this regard, the authors speculate that it could be possible that a prolonged period of exposure might be necessary to alter the P53 pathway that is involved in the progression of NMIBC to MIBC. It would be therefore be useful to specifically investigate the mechanisms and the alterations necessary for this to happen, especially on a structural basis like LOH and copy number alterations. Detailed molecular studies using this cellular model will eventually help to identify related genes and pathways that are altered due to smoking in a stepwise fashion. Ultimately, accumulated knowledge will help to develop personalized management of UCC patients.

Brait M, Munari E, Lebron C, Noordhuis MG, Begum S, Michailidi C, et al. Genome-wide methylation profiling and the PI3K-AKT pathway analysis associated with smoking in urothelial cell carcinoma. Cell Cycle. 2013;12:1058–70. doi: 10.4161/cc.24050.